1. Field of Invention
This invention concerns a new and effective method and assay for determination of functionality of steroid receptors present in breast or other tumors. The method determines cancer responsiveness to hormonal therapy by measuring the level of DNA-binding (functioning) and non-DNA binding (nonfunctioning) steroid receptors and establishing a correlation between such functioning estrogen receptors and those which are dysfunctional or nonfunctional. In particular, this method distinguishes and quantifies steroid receptors capable of binding to DNA from those which do not bind to DNA or bind abnormally. The two-step gel-shift Western blot assay is useful for determination whether the cancer, particularly the breast cancer, will respond to anti-estrogen hormonal therapy.
2. Background Art and Related Disclosures
Breast, uterine or prostate cancers are among the most serious cancerous diseases. Breast carcinoma is one of the most common malignancy among women and has the one of the highest fatality rate of all cancers affecting this sex. American Cancer Society estimates that around 44,000 U.S. women die from breast cancer each year.
Treatment of the breast, ovarian, uterine or prostate cancer may be either palliative or curative. The choice of treatment depends mainly on the extent of the disease. When the cancer is localized primarily in the breast, ovarian, uterine or prostate tissue, treatment by surgery is usually the first choice therapy. Radiotherapy, hormonal therapy, cytotoxic chemotherapy and to a lesser degree immunotherapy are used for both the palliative treatment or as adjuvants to surgery. Both radiotherapy and chemotherapy are accompanied by many unpleasant, undesirable and dangerous side effects. Immunotherapy is still in its experimental stage.
For the past 15 years, hormonal therapy had been shown to be effective, to a certain degree, for breast, uterine or for prostate cancer treatment. Hormonal therapy is typically used in palliation of symptoms or in delaying advance of the disease. Hormonal therapy utilizes analoques of both androgens and estrogens, however, in patients having postmenopausal or recurrent breast cancer, estrogen-based treatment is preferred.
In recent years, in pursuance of research related to breast cancer and its treatment, the presence of so called estrogen-receptors (ER) and progesterone-receptors (PR) was discovered in certain breast tumors. Currently, it is believed that women whose breast cancer cells contain estrogen and/or progesterone receptors have a much better chance to survive if they are treated with estrogen blocking drugs such as Tamoxifen, a non-steroidal estrogen antagonist.
The presence or absence of estrogen and progesterone-receptor protein in primary or metastatic tumor tissue is used nowadays to predict which patient may be expected to respond to additive or ablative endocrine therapy. The presence of ER is correlated with expected responsivity of the cancer to the hormonal therapy. The absence of ER is correlated with expected nonresponsiveness to the hormonal therapy. Notwithstanding, some 20-70% of tumors containing estrogen receptors (ER) fail to respond to hormone therapy. This leads to a belief that some of these estrogen receptors may be nonfunctional or dysfunctional in some way. Since the untreated breast cancer is a fatal disease, such non-responsiveness to the hormonal therapy due to the estrogen receptors dysfunctionality has very serious consequences. Patients with tumors known to contain abnormal ER where the probability of the cancer responsiveness to hormonal treatment is low would be offered other forms of therapy instead of hormonal therapy.
It would, therefore, be of a great prognostic value and therapeutic importance to have available a method and assay for determination whether the estrogen receptors are functioning, what is the correlation of the functional to nonfunctional receptors and to predict from such determination whether the particular cancer cells containing estrogen receptors will respond to a hormonal therapy.
Estrogen receptors belong to a family of nuclear receptors whose function is dependent on the binding of small hydrophobic ligands. In the estrogen receptor case, it depends on the binding of the hormone estradiol. The binding of estrogen receptors (ER) to its estrogen responsive element (ERE) has been described in Cell, 55:145 (1988). Estradiol or the anti-estrogen hydroxytamoxifen were shown to be necessary for the estrogen receptor binding to its ERE binding domain on DNA.
Gene transfer studies have shown that estrogen regulation of specific genes is mediated by ERE. This binding interaction is highly sequence and receptor specific. Estrogen receptor seems to bind to the ERE as a head-to-head dimer. The binding of estrogen receptor to the ERE is detectable by a gel retardation (band shift) assay. Such assay, utilizing ERE of the vitellogenin A2 gene for specific estrogen receptor binding, is described in Cel. Biol., 9:43 (1989). The presence of estrogen receptors in the receptor protein-DNA complexes is measured by mobility shift of the protein-DNA complexes. The specificity of the ERE for estrogen receptor is evidenced by the lack of estrogen binding to a progesterone response elements (PRE).
The first modifications to a basic gel-shift assay described above were disclosed by inventors during the 12th Annual San Antonio Breast Cancer Symposium, held on Dec. 8-9, 1989, and are briefly and incompletely described in the Proceedings Abstracts, published in Breast Cancer Res. Treat., 14:178 (1989). The modified gel-shift assay determines the formation of ER-ERE complexes as a measure of ER activation and functionality which is necessary for a responsiveness of the breast cancer to hormonal treatments.
Previously used receptor assay techniques were directed only toward a determination of the presence or the absence of ER and its quantification but not to the activation and functionality of ER which is believed to be necessary for both high-affinity DNA (ERE) binding and for a transcriptional control. The presence of ER in the breast cancer cells that is unable to bind to ERE, or binds to ERE in an improper form may thus explain prior clinical findings of ER-positive and yet hormone resistant breast cancers.
The primary feature of this invention is thus directed toward a method and ultimately toward an assay which measures both the ability to form ER-ERE complexes and the amount of ER that is unable to bind ERE, both of which determine ER functionality. Ultimately, the utility of this invention lies in a determination whether breast cancer will respond to hormonal treatment or not.
The one-step gel-shift DNA binding receptor assay suitable to distinguish between functional and nonfunctional ER-ERE isoforms is disclosed herein as one novel feature of this invention. The second feature of this invention is directed toward a two-step gel-shift Western blot assay which can distinguish and quantify functioning steroid receptors present in human tissue i.e., distinguish those receptors which are capable of binding normally or abnormally to DNA by gel-shift assay, from those receptors which are nonfunctional, i.e. which fail to bind to DNA (ERE) completely. For this purpose, the new two-step gel-shift Western blot assay for detection and quantitation of non-DNA binding steroid receptors has been developed and is disclosed herein as yet another novel feature of this invention.